Targeting cd3 for immune modulation with rna aptamers

Resumen

Targeting CD3 for immune modulation with RNA Aptamers To identify aptamers capable of eliciting T cell activation by providing agonistic signals to the murine CD3 complex, we performed HT-SELEX against the recombinant mouse delta epsilon heterodimeric protein using a 2’ Fluoropyrimidine RNA library. After six rounds of selection, numerous aptamer families were enriched, out of which four aptamers were selected for further characterization based on their ability to specifically bind to target protein. These aptamers were found to bind to CD3 expressing EL-4 Cells, as well as primary CD8 T cells, and the specificity of their binding was confirmed using CD3KO cell lines. Aptamers 1 and 12 induced calcium flux in T cells and lowered the threshold of their activation, especially in the presence of suboptimal pMHC stimulation. Upon dimerization, CD3δε aptamers enhanced proliferation of CD4 and CD8 T cells receiving suboptimal polyclonal CD3 antibody stimulus. Furthermore, they also enhanced proliferation of low affinity and high affinity tumor reactive T cells suboptimally activated with their cognate antigens. In vivo, CD3δε aptamers enhanced immune responses generated by cancer antigen vaccines. Enhanced proliferation and IFN-ɣ producing ability was observed in splenocytes of vaccinated mice, and tumor growth was reduced in vivo. Even though intratumoral treatment with CD3δε aptamer did not protect against tumor growth, adoptive transfer of tumor reactive T cells expanded in the presence of CD3δε aptamers could generate anti-tumor immunity that slowed growth of established aggressive melanoma tumors in mice. Lastly, this aptamer shows cross reactivity to the human CD3 protein complex at the protein and cellular levels, increasing its potential for clinical translation if functional characterization with human T cell models is successful. Thus, this study highlights the development of a novel off-the-shelf RNA aptamer therapeutic that elicits T cell activation, proliferation and enhanced effector function in a CD3 specific manner.